Journal of Oleo Science Copyright ©2015 by Japan Oil Chemists’ Society doi : 10.5650/jos.ess14177 J. Oleo Sci. 64, (2) 223-232 (2015)
Analysis and Antibacterial Activity of Nigella sativa Essential Oil Formulated in Microemulsion System Hamdy A. Shaaban1, Zainab Sadek2, Amr E. Edris1＊ and Amal Saad-Hussein3 1
Aroma & Flavor Chemistry Department, National Research Center, Cairo, Egypt Dairy Department, National Research Center, Cairo, Egypt 3 Environmental & Occupational Medicine Department National Research Center, Cairo, Egypt 2
Abstract: The Essential oil (EO) of Nigella sativa (black cumin) was extracted from the crude oil and the volatile constituents were characterized using gas chromatographic analysis. The EO was formulated in water-based microemulsion system and its antibacterial activity against six pathogenic bacteria was evaluated using the agar well diffusion method. This activity was compared with two other well known biologically active natural and synthetic antimicrobials namely eugenol and Ceftriaxone®. Results showed that N. sativa EO microemulsion was highly effective against S. aureus, B. cereus and S. typhimurium even at the lowest tested concentration of that EO in the microemulsion (100.0 μg/well). Interestingly, the EO microemulsion showed higher antibacterial activity than Ceftriaxone solution against S. typhimurium at 400.0 μg/well and almost comparable activity against E. coli at 500.0 μg/well. No activity was detected for the EO microemulsion against L. monocytogenes and P. aeruginosa. Eugenol which was also formulated in microemulsion was less effective than N. sativa EO microemulsion except against P. aeruginosa. The synthetic antibiotic (Ceftriaxone) was effective against most of the six tested bacterial strains. This work is the first report revealing the formulation of N. sativa EO in microemulsion system and investigating its antibacterial activity. The results may offer potential application of that water-based microemulsion in controlling the prevalence of some pathogenic bacteria. Key words: antibacterial activity, Nigella sativa, essential oil, eugenol, microemulsion, Ceftriaxone 1 INTRODUCTION Essential oils（EOs） are natural mixtures of volatile compounds secreted as by-products by many aromatic and medicinal plants. These oils act as insect attracting agents for pollination and also as a defense line against microbial attack. EOs were first used as perfuming and flavoring agents then their inherent antimicrobial activity was discovered. Different investigations on the application of EOs against pathogenic bacteria, food spoilage and film forming microorganisms have been reported1−4）. The antimicrobial properties of EOs against pathogens were studied in different sanitizing applications including sanitization of wounds5） and surfaces6）, disinfection of animal houses7） and sewage grey water8）. The promising results of these investigations along with many others warranted EOs for consideration as potential candidates for application as adjuvant for the synthetic antibiotics. EOs can enhance the therapeutic efficiency of these antibiotics through a synergistic mechanism9, 10）. Combination of antibiotics with some
EOs can also reduce the well known antibiotic resistance in multidrug resistant bacteria11）. Interestingly, it was found that formulation of EOs as nanoparticles in water-based delivery systems can enhance their antimicrobial activity compared with the neat unformulated form of these oils12, 13）. The mechanisms that clarify the role of nanoparticle in boosting the antimicrobial activity of EOs were reviewed in details14）. The above mentioned literature included antimicrobial evaluations of wide varieties of EOs isolated from different aromatic plants. However, we noticed some limitation of conclusive data regarding the antibacterial activity of Nigella sativa EO especially after formulation in microemulsion. The seeds of that herbaceous plant（family Ranunculaceae）are the part that bears the EO. The antimicrobial evaluation of the seeds and their solvent extracts were studied15−18）. Specific targeting on the antimicrobial evaluation of the pure EO fraction of N. sativa has also been approached19）. However that study did not address
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Correspondence to: Amr E. Edris, Aroma & Flavor Chemistry Department National Research Center El Behose Street, Dokki, 12622 Cairo, Egypt E-mail: [email protected] Accepted September 27, 2014 (received for review August 10, 2014)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
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H. A. Shaaban, Z. Sadek, A. E. Edris et al.
some important issues that usually correlated with the antimicrobial activity of N. sativa EO. These include the chemical composition and the percentage of the major volatile constituents of that EO especially thymoquinone （TQ） . On the other hand, some other antimicrobial evaluations considered and correlated the chemical composition of the EO to the reported activity20−22）. However, these investigations did not include some important pathogenic bacteria in their evaluations like Bacillus cereus, Listeria monocytogenes, and Salmonella typhimurium. Most importantly, no studies were found, to our knowledge, evaluating the antibacterial activity of N. sativa EO as nanoparticles formulated in water-based microemulsion delivery system. From the above mentioned, the following investigation was dedicated to provide more information on the antibacterial activity of that EO microemulsion which may not be addressed in previous studies. This included extraction and analysis of the EO followed by formulation in waterbased microemulsion system and finally evaluation of the overall antibacterial activity of the formulated EO against different pathogenic bacteria. Moreover, the antibacterial activity of the EO microemulsion was compared with that of a well know plant-based phenolic antimicrobial model namely eugenol which was formulated also in microemulsion. A synthetic antibiotic model （Ceftriaxone） was used as a reference to help in ranking the antimicrobial activity of N. sativa EO microemulsion among the natural（eugenol） and the synthetic （Ceftriaxone） antimicrobial models.
2 EXPERIMENTAL 2.1 Materials Packages of N. sativa crude oil encapsulated in hard gelatin capsules were purchased from a local pharmacy in Cairo, Egypt. Each capsule contains 450.0 mg of a 100.0％ pure crude oil extracted from the seeds of N. sativa by cold expression. No other additives are included in the capsules, as indicated by the manufacturing company （Pharco Pharmaceuticals, Cairo, Egypt） . The synthetic antibiotic Ceftriaxone® sodium powder （containing 1000 mg Ceftriaxone） was also purchased from local pharmacy in Cairo, Egypt. The drug is produced by SmithKline Beecham Egypt under license from Sandoz. ® sorbitane Eugenol 99.0％ and Tween 20（polyoxyethylene mono-laurate）were purchased from Sigma-Aldrich Chemical Co. （St. Louis, MO, USA） 2.2 Extraction of the pure EO fraction Sufficient numbers of capsules bearing N. sativa whole crude oil（which is a natural mixture of fixed oil and EO）, were broken and evacuated into a beaker. The pure EO fraction was extracted from the crude oil by the hydro-dis-
tillation method as previously indicated 23）. In brief, the crude oil was mixed with distilled water（1:5 w/v）and distilled for three hours using Clevenger-type apparatus. The cup that receives the pure EO fraction in the side arm of the apparatus was wrapped with aluminum foil to protect the EO from light that can dimerize its main constituent thymoquinone into di-thymoquinone. At the end of the distillation process, the EO was collected, weighed and its content（yield percent）relative to the weight of total crude oil was calculated as mean of two extraction processes. 2.3 Gas chromatographic analysis （20.0 μl） was diluted in 1.0 ml diethyl ether N. sativa EO in a glass vial. Then 2.0 μl of this mixture were injected （at a split ratio 10:1）into a Perkin Elmer XL GC equipped with a FID. A fused silica capillary column（60 m×0.32 mm× 0.25 μm）coated with DB-5（5％ phenyl, 95％ methyl polysiloxane）was used to separate the different EO components. The oven temperature was programmed from 50℃ to 230℃ at a rate of 3℃/min. The injector and detector temperatures were 230℃ and 250℃ respectively. Helium was used as carrier gas at a flow rate of 1.0 ml/min. The volatile oil constituents were quantified as percent of the total detected peak areas after flame ionization detection. All values were mean of two injections from two different extractions±SD. The available authentic samples were used to reveal the identity of N. sativa EO constituents by matching their retention times with those of N. sativa EO constituents after running on GC under the same conditions. 2.4 Gas chromatographic-mass spectroscopic analysis GC-MS analysis was conducted using a Hewlett-Packard 5972 GC-MS system equipped with the same column and conditions used as in GC analysis. The ionization voltage was 70 eV and the ion source temperature was 170℃. The scan range was from 29 to 300 AMU at 2.76 scans per second. Components identity that was previously revealed by running authentic samples in GC analysis was verified by automatic matching of their fragmentation patterns（m/ z）with standard components stored in the electronic mass spectral library（NIST: National Institute of standards and Technology）that was built-in the GC-MS soft ware. 2.5 Formulation of N. sativa EO and eugenol in waterbased microemulsions Surfactant solution was prepared by dissolving the nonionic surfactant（Tween 20®）in de-ionized water at 5.0％ （w/w）using magnetic bar. The clear solution was divided equally by weight among different glass vials wrapped with aluminum foil to protect from light. Different volumes of the hydrophobic N. sativa EO and eugenol were titrated separately into each vial to end up with 10 concentrations for both EO and eugenol, ranging between 0.2％ - 2.0％
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Analysis and Antibacterial Activity of Nigella sativa Essential Oil Formulated in Microemulsion System
with a concentration gradient of 0.2％. This percentage was equivalent to 2.0 mg - 20.0 mg EO or eugenol/ml microemulsion with concentration gradient 2.0 mg. All vials were vortexed for 1 minute to mix the ingredients and hasten the formation of microemulsion then left in dark at 27℃±1 to equilibrate. All formulations were also made in duplicates. 2.6 Formulation of Ceftriaxone in aqueous solution Unlike the hydrophobic EO and eugenol, Ceftriaxone ® hydrophilic powder was re-constituted directly in distilled water to give 1.0％ aqueous solution（i.e. 10 mg/1 ml water）. Then serial dilutions were made from that stock solution as required for utilization in the antibacterial assay. Thus there was no need to formulate Ceftriaxone in water-based microemulsion as it is a water-soluble antimicrobial. 2.7 Verification of microemulsion formation Evidence of microemulsion formation for N. sativa and eugenol was based on the clear and transparent appearance of the formulations after the end of equilibration time which reached 4 weeks. The non-birefringent texture under polarized light microscope and the small hydrodynamic particle size（r）which did not exceed 100.0 nm was also taken as an evidence of microemulsion formation. 2.8 Particle size measurement The particle size of microemulsions was measured using the dynamic light scattering instrument Zetasizer （Nano-ZS model ZEN3600, Nanoseries, Malvern Instruments, UK）. Measurements were done at 27℃, with a fixed angle of 173° . The measurements are based on the Brownian motion of the hydrated particles, thus it provides information on the hydrodynamic diameter（nm）of the microemulsion particles. Sizes quoted are the z-average mean of the microemulsion hydrodynamic diameter（nm）obtained from 10 measurements （2 replicate×5 measurements each） . Before measurement, the samples were filtered through 0.20 μm single use syringe filter unit（Minisart®, Sartoius Stedium Biotech GmbH Germany） to remove impurities. 2.9 Antibacterial evaluation 2.9.1 Microorganisms and cultures The tested microorganisms were provided from the culture collections of the Microbiological Department National Research Center（NRC）Dokki, Giza, Egypt. These include three strains of Gram-positive bacteria Staphylococcus aureus（ ATCC 43300）, Bacillus cereus（ ATCC 11778） , Listeria monocytogenes （ATCC 35152） , and three strains of Gram-negative bacteria Salmonella typhimuri（ATCC 13311） , Escherichia coli （ATCC 27325） , Pseuum domonas aeruginosa （ATCC 27853） .
2.9.2 Antibacterial assay The antimicrobials N. sativa EO, eugenol and Ceftriaxone formulated in their corresponding delivery systems （see formulation section）were evaluated against the six previously mentioned pathogenic bacteria. The evaluation was performed at five chosen concentrations of the three antimicrobials: 0.2％, 0.4％, 0.6％, 0.8％ and 1.0％, unless otherwise stated. These percentages are equivalent to 2.0 mg antimicrobial/1.0 ml formula, 4.0 mg/ml, 6.0 mg/ml, 8.0 mg/ml and 10.0 mg/ml, respectively. The agar well diffusion method24） was employed for the determination of antibacterial activities of the three formulated antimicrobials. In details, 0.1 ml of the diluted inoculums（107 CFU/ml） of test organism was spread on tryptone soy agar（TSA） plates. Wells of 5 mm diameter were punched into the agar medium and filled with 50 μl of each concentration of the three evaluated antimicrobial formulas. This volume delivered 100.0 μg, 200.0 μg, 300.0 μg, 400.0 μg and 500.0 μg of each antimicrobial（EO, eugenol and Ceftriaxone）per well. The plates were incubated for 18 h at 37℃. Antimicrobial activity was evaluated by measuring the zone of inhibition （mm）against the tested organisms. The evaluation was conducted twice and each time comprised three replicates for each concentration. 2.10 Statistical analysis Statistical analysis was done through SPSS version 18.0. Quantitative data were represented in form of mean±standard deviation（ SD）. Analysis of Variance（ ANOVA）was used in the analysis of the results. The post hoc Duncan was used to make pair wise comparisons using a stepwise order of comparisons identical to the order used by the Student-Newman-Keuls test, but sets a protection level for the error rate for the collection of tests, rather than an error rate for individual tests. The level of significant was considered at p＜0.05.
3 Results and Discussions 3.1 Analysis of N. sativa EO The content of the pure N. sativa EO fraction after extraction from the whole crude oil using the hydro-distillation method was 2.2±0.1 wt ％. This percentage is considered to be much higher than other samples of N. sativa that yield 0.11-1.8 wt ％ EO relative to the crude oil23）. The genetic diversity of the seeds is among the most important factors that affect the quality and content of the EO25）. Table 1 showed the main constituents of the EO as re（TQ） vealed by the GC and GC-MS analysis. Thymoquinone was the major constituent of the EO（52.6％）followed by p-cymene（25.8％）, α-thujene（10.5％）, β-pinene（3.0％） and α-pinene（2.8％）. Other minor constituents were also 225
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Table 1 Main constituents of N. sativa EO as revealed by GC and GC-MS analysis. Retention time (min)
Area %#
α-thujene*
Component
14.3
10.50±0.7
93 (100.0%), 77, 91, 92, 41, 39, 27, 79, 94, 136
α-pinene*
14.6
2.8 ±0.2
93 (100.0%), 91, 92, 39, 77, 41, 79, 27, 53, 29
sabinene
16.3
0.5 ±0.07
93 (100.0%), 41, 91, 77, 79, 39, 27, 69, 94, 43
β-pinene*
16.6
3.0 ±0.3
93 (100.0%), 41, 69, 39, 91, 77, 79, 27, 92, 53
O-cymene
18.3
0.1 ±0.02
119(100.0%), 91, 134, 117, 77, 65, 115, 39, 120, 51
p-cymene*
18.7
25.8 ±0.7
limonene*
19.0
1.1 ±0.1
68 (100.0%), 93, 67, 94, 39, 92, 107, 53, 79, 136
linalool*
22.3
0.5 ±0.05
71 (100.0%), 93, 55, 43, 41, 69, 80, 121, 67, 39
iso-3-thujanol
22.9
1.36±0.2
43 (100.0%), 55, 41, 93, 95, 81, 67, 79, 121, 69
thymoquinone*
29.6
52.6 ±1.9
α –longipinene
33.4
0.3 ±0.03
119 (100.0%), 105, 133, 93, 107, 91, 161, 120, 204, 121
longifolene
35.8
1.5 ±0.1
161 (100.0%), 94, 91, 93, 107, 105, 95, 79, 55, 109
Total
Mass fractions of each component (m/z)**
119(100.0%), 134, 91,120, 117, 41, 77, 39, 65, 115
164 (100.0%), 121, 136, 93, 149, 39, 40, 53, 77, 91
98.96
#: after GC analysis via flam ionization detection * Component identity was confirmed by matching retention times with authentic samples, beside the GC-MS identification. ** Mass fractions of each component were arranged in descending order according to the % abundance. identified and illustrated in the table. This composition authenticated and confirmed the identity of N. sativa crude oil in the gelatin capsules because high p-cymene and TQ percentages in the EO are considered as markers for the sativa species. p-cymene and TQ can reach only 0.0-0.5％ and 0.0-0.1％, respectively in the EO of other Nigella species like for instance Nigella orientalis, N. damascene and N. arvensis26−28）. 3.2 Formulation of microemulsions After the characterization of N. sativa EO, five concentrations of this oil and the pure eugenol were formulated separately in water-based microemulsion systems with the aid of surfactant（Tween 20）. Micro-emulsification technique is simple and economically feasible on small as well as large scale for solubilizing hydrophobic bioactive oils in water-based environment as nano-particles. This process is spontaneous and driven by the reduction of the system’ s free energy due to interaction of the hydrophobic solubilizates （N. sativa EO and eugenol） with the surfactant molecules at certain energetically favorable orientation. One should consider that formulation of both EO and eugenol in water-based micoemulsion is necessary due to the hydrophobic nature of these antimicrobials which prevent direct dissolution in water. On the other hand, the synthetic antibiotic Ceftriaxone was re-constituted directly in
Fig. 1 P article size distribution of Nigella EO and eugenol microemulsions measured at their maximum concentrations (1.0% and 0.9%), respectively. water due to its hydrophilic nature. Figure 1 showed that the particle size of the EO and eugenol in the microemulsion formulations was 8.7±0.1 nm and 8.9±0.1 nm, respectively with mono-modal size distribution. Examination of the different formulation under the polarized light microscope revealed no birefringence. Visual observation showed clear and transparent
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Analysis and Antibacterial Activity of Nigella sativa Essential Oil Formulated in Microemulsion System
Fig. 2 C hemical structures of thymoquinone and eugenol. formulation with water-like flowability. These data confirmed the formation of typical water-based microemulsion systems. Both microemulsions were found to be physically stable when stored at 37℃ for 24.0h before the evaluation of their antibacterial activity. They were also physically stable towards different freeze-thaw cycles and toward centrifugation at 5000 rpm for 30 min which confirmed their kinetic and thermodynamic stability as microemulsions. It was interesting to note during formulation that eugenol took only ~ 30.0 minutes to equilibrate into a clear transparent microemulsion while N. sativa EO needed longer time that can reach 4.0 week to equilibrate especially at high concentration like 1.0％（i.e. 10.0 mg EO/ml microemulsion）. The reason of that observation lies in the chemical structure of eugenol that contains hydrophilic and hydrophobic-like parts resembling the surfactant structure（ Fig. 2）. Due to that structure, eugenol can arrange itself at the interfacial film along with the original surfactant（Tween 20）. That arrangement makes the original surfactant more efficient and facilitates the formation of microemulsion29）. That advantage is not available for N. sativa EO which is a complex mixture of volatile components of different chemical structures and polarities. One of the liabilities of microemulsion formulation is the low pay load of the bioactive oil that can be incorporated in the water-surfactant solution to form microemulsion. Thus it was important to determine the maximum oil concentration that can be incorporated into the microemulsion before evaluating the antibacterial activity. For N. sativa EO that concentration was found to be 1.4 ％（i.e. 14 mg EO/ ml microemulsion）under the formulation conditions adopted in the current study. However at that concentration, crystals of TQ, which is the major constituent of the EO（52.6％）, were precipitated out from the microemulsion. Therefore the upper limit of N. sativa EO used for assessing the antibacterial activity in the microemulsion was set at 1.0％ （10 mg EO/ml microemulsion, or 500.0 μg/ well）. This concentration proved to be the safe margin for keeping the integrity of the EO constituents （including TQ） as being totally incorporated in the microemulsion. On the
other hand concerning eugenol, the maximum pay load in the microemulsion was found to be 0.9％（9.0 mg eugenol/ under the same formulation conditions. ml microemulsion） Trials to increase that load to reach 1.0％ in order to match that of N. sativa EO was unsuccessful as it turned the transparent eugenol microemulsion into a cloudy dispersion of macroemulsion. The particle size of eugenol in that macroemulsion was out of the microemulsion size range （＞ 100 nm）as revealed by the visual cloudy appearance and the presence of large particles under the light microscopic. 3.3 Antimicrobial evaluation Table 2 showed the antimicrobial activity of the three tested formulas namely, EO microemulsion, eugenol microemulsion and Ceftriaxone solution against some pathogenic bacteria. The table showed that the EO microemulsion was more antibacterial active than eugenol microemulsion at all concentrations against S. aureus, B. cereus and S. typhimurium, while eugenol was more effective against P. aeruginosa. The activity of eugenol microemulsion against P. aeruginosa reached its climax at 500.00 μg/well at which there was no significant difference between the antibacterial activity of eugenol microemulsion（40.0 mm）and that of Ceftriaxone（40.3 mm）. Interestingly, there was no significant difference between the antimicrobial activity between N. sativa EO microemulsion（18.0 mm）and Ceftriaxone（19.3 mm）against S. typhimurium at 300.0 μg/ well. In addition, the EO microemulsion showed even higher antibacterial activity than Ceftriaxone against S. typhimurium at 400.0 μg and 500.0 μg. The EO microemulsion also showed close antimicrobial activity（30.3 mm）to Ceftriaxone（31.3 mm）against E. coli at 500.0 μg. From Table 2 it is evident that the EO microemulsion of N. sativa at high concentrations（400.0 μg/well and 500.00 μg/well）possesses antibacterial activity against four out of six of the tested pathogens. The EO microemulsion showed its highest antibacterial activity at the lowest concentration （100.0 μg EO/well）against S. aureus and B. cereus, respectively. E. coli was more resistant to EO microemulsion up to 300.0 μg/well, and then as the concentration of EO increased to 400.0 μg/well the antimicrobial activity also increased until reaching maximum（30.3 mm）at 500.0 μg/ well. On the contrary, L. monocytogenes and P. aeruginosa were the most resistant bacterial strains toward EO microemulsion treatments at all concentrations. The highest antimicrobial activity for EO microemulsion folat 500.0 μg/well was found against S. aureus（33.7 mm） lowed by E. coli（30.3 mm）, S. typhimurium（26.5 mm） and B. cereus（21.3 mm）, respectively. Table 2 also showed that eugenol microemulsion possessed high antibacterial activity against P. aeruginosa at all concentrations. Eugenol microemulsion was not effective at all concentrations against B. cereus and L. monocytogenes. However, it started to show concentration depen227
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Table 2 Effect of the three tested antibacterial formulas against some pathogenic bacteria. Concentration*
Antibacterial formulas EO microemulsion
100.0 μg/well
Eugenol microemulsion Ceftriaxone solution P-value EO microemulsion
200.0 μg/well
Eugenol microemulsion Ceftriaxone solution P-value EO microemulsion
300.0 μg/well
Eugenol microemulsion Ceftriaxone solution P-value EO microemulsion
400.0 μg/well
Eugenol microemulsion Ceftriaxone solution P-value
500.0 μg/well
S. aureus
E. coli
B. cereus
L.monocytogenes
P. aeruginosa
S. typhimurium
Inhibition zone (mm) Mean
SD
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Mean
SD
15.7
0.6
0.0a
0.0
14.7
0.6
0.0a
0.0
0.0
0.0
8.0
1.7
0.0
0.0
0.0a
0.0
0.0
0.0
0.0a
0.0
16.0
1.0
0.0
0.0
39.67
0.6
24.0
1.0
27.0
1.7
16.7
1.5
34.0
1.7
16.7
1.5
P< 0.0001
P< 0.0001 a
P< 0.0001
P< 0.0001 a
P< 0.0001
P< 0.0001
19.7
2.1
0.0
0.0
16.3
1.5
0.0
0.0
0.0
0.0
16.0
0.0
0.0
0.0a
0.0
0.0
0.0
0.0a
0.0
21.3
1.5
0.0
0.0
41.7
0.6
25.7
1.2
28.3
1.5
17.7
1.5
36.0
1.0
18.0
1.0
P< 0.0001
P< 0.0001
2.3
0.0a
0.0
0.0
a
0.0
0.0
43.3
0.6
27.3
0.6
25.3
P< 0.0001
0.0
P< 0.0001
P< 0.0001
P< 0.0001
1.0
0.0a
0.0
0.0
0.0
a
29.7
1.2
18.7
18.0
P< 0.0001
0.0
P< 0.0001 0.0
0.0
1.0
P< 0.0001 18.0a
1.7
0.0
26.0
1.4
0.0
0.0
1.5
37.3
0.6
19.3a
1.2
P< 0.0001
P< 0.0001
P< 0.0001
33.0
1.0
25.3
1.5
19.6
1.5
0.0a
0.0
0.0
0.0
22.3
0.6
7.7
0.6
13.5
0.7
0.0
0.0
0.0a
0.0
32.7
1.2
12.3
0.6
44.7
0.6
29.0
1.0
32.0
1.0
20.0
1.0
39.3
0.6
21.7
1.2
P< 0.0001
P< 0.0001
P< 0.0001
P< 0.0001
P< 0.0001
P< 0.0001
EO microemulsion
33.7
1.5
30.3
1.5
21.3
1.5
0.0a
0.0
0.0
0.0
26.5
0.7
Eugenol microemulsion
12.7
0.6
15.7
1.2
0.0
0.0
0.0a
0.0
40.0a
1.0
16.3
0.6
Ceftriaxone solution
46.0
1.0
31.3
1.6
34.7
0.6
21.0
2.0
40.3a
0.6
23.7
2.1
P-value
P< 0.0001
P< 0.0001
P< 0.0001
P< 0.0001
P< 0.0001
P< 0.0001
* The content of the antimicrobial agent (μg) which present in 50.0 μl of each antimicrobial formula /well a : Means with the same letter are not significantly different. The highest tested concentration of eugenol in the microemulsion was 0.9% (450 μg/well) due to formulation restriction (as indicated previously in the results and discussion section).
dent antibacterial activities above 300.0 μg/well against S. aureus, E. coli and S. typhimurium. The inhibition zone of P. aeruginosa was still significantly larger than that of these three strains at concentration above 300.0 μg/well. From the table it is also clear that Ceftriaxone solution had high antibacterial active against all bacterial strains at all concentrations. However, it was more effective against S. aureus and P. aeruginosa compared with L. monocytogenes and S. typhimurium at the lowest and highest tested concentrations. It is impotent to note that the control sample of the surfactant and water at 5.0％ did not show any antibacterial activities. These results in general indicated a selective and high antibacterial activity of N. sativa EO. This activity is thought to be due to the high content of TQ which constitutes 52.6％ of the EO composition（Table 1）. This compound is a phyto-quinone present only in the EO of N. sativa. One of the most important antimicrobial characteristics of TQ is its ability to modify the resistance of bacteria to antibiotics by acting as efflux pump inhibitor30）. Efflux pumps are proteins in the cell membrane of the bacteria that extrude the antimicrobial agents outside the cell31）. Thus multi-drug resistant bacterial strains can arise 32）
which threaten the lives of patients infected with these strains. TQ was efficient in inhibiting several multi-drug resistant pathogenic bacteria19）. It can kill antibiotic resistant S. aureus at 6.0 μg/ml33）. TQ also has anti-biofilm properties against some Gram-positive and Gram-negative bacteria34）. Beside literature evidences that points out to TQ as the major antibacterial active principle of N. sativa EO one should bear in mind the potential synergistic effect of TQ with other component（s）that constitute the EO. For instance, p-cymene which constitutes 25.8％ of the EO composition may act as a potential synergistic agent. This terpenic hydrocarbon was previously found to act synergistically with carvacrol against B. cereus in rice35） and against Escherichia coli O157:H7 in un-pasteurised apple Juice36）. p-Cymene also showed a high affinity for microbial membranes however it does not affect the membrane permeability but it may decrease its enthalpy and melting temperature37）. In addition, this compound can perturb the microbial membranes, causing them to expand thus affecting the membrane potential of the intact cells38）. Synergism can also be rationalized in some cases by the protection of the major antimicrobial principle of a given EO from degradation by the microbial enzymes or by con-
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tribution in altering the multi-drug resistance mechanism39）. To our knowledge, there is no data so far revealing the potential synergistic activity between p-cymene and TQ which could be a subject for a future research work. Despite this evidence-based antibacterial activity of N. sativa EO microemulsion, it was challenging to find it not effective against L. monocytogenes and P. aeruginosa at all concentrations （Table 2） . The relatively simple structure of the cell wall of L. monocytogenes as Gram-positive bacteria is supposed to make it vulnerable to a wide range of antibacterial agents. However the antimicrobial resistance of L. monocytogenes is not an unusual biological event. This phenomena was previously reported in different studies from samples taken from dairy farms 40）, seafood products41）, and from ready-to-eat meat products42）. The resistance of L. monocytogenes originates from some antimicrobial resistance genes that limit the number of the effective anti-listerial agents, as revealed from the last three references. Details of the resistance mechanism of L. monocytogenes were reviewed elsewhere43）. P. aeruginosa was found to be another resistant bacterium to N. sativa EO microemulsion. The complex cell envelope of this organism resembles that of other Gram-negative bacteria which consist of polysaccharide, protein, lipopolysaccharide, and lipid components 44）. It is well known that P. aeruginosa possessed a great adaptability and metabolic versatility. It has adaptive resistance toward some antibiotics45, 46） and biocides47）. Adaptive resistance refers to reversible refractoriness to the bactericidal action of an antibiotic following first exposure. This phenomenon has been well documented in P. aeruginosa following exposure to an aminoglycoside48）. Thus the potential reasons for resistance to antimicrobials are a combination between the inherent multidrug efflux pumps with chromosomallyencoded antibiotic resistance genes49）and the barrier function of the outer complex cell membrane. Another acquired resistance factor may also develop by mutation in chromosomally-encoded antibiotic resistance genes50）. A detailed contribution explaining the mechanism of antibiotic resistance in P. aeruginosa is also available elsewhere51）. Interestingly, with the presence of all the above mentioned resistance mechanisms against antibacterial agents, eugenol microemulsion was found to be highly effective against P. aeruginosa than N. sativa EO at all concentrations（ Table 2）. The activity of eugenol microemulsion against that pathogen was almost the same as that of Ceftriaxone at the highest concentration（ 500.0 μg/well）. Another study also reported high antibacterial activity of clove bud ethanolic extract, as a rich source of eugenol, against P. aeruginosa52）. However, the incidence of antimicrobial sensitivity for that organism varied depending on the isolate strain and source of isolation （human, animal or environment）. This may explain why two strains of P. aeruginosa exhibited intrinsic tolerance to different plant-
based volatiles including pure eugenol53）. The disrupting effect of eugenol on the cytoplasmic membrane of the bacterial cell54） may justify its observed effectiveness against P. aeruginosa in our study. Another reason may include a certain affinity between the surfactant（Tween 20）that associate with the active phenolic group of eugenol in the microemulsion, and the cell membrance of P. aeruginosa. That affinity can induce better adsorption of the surfactant/eugenol complex to the surface of the bacterial cell which in turn will increase its local concentration at that point. However, a conclusive justification of the enhanced antibacterial activity of eugenol microemulsion against P. aeruginosa in our study is certainly challenging. That is particularly due to the lack of any antibacterial activity of eugenol microemulsion against L. monocytogenes and B. cereus at all concentrations（Table 2） . In contrast to this result, eugenol microemulsion was found in other studies effective against L. monocytogenes at concentrations close to that used in the current study55−57）. This contradiction may originate from using different strain or isolate of L. monocytogenes that lack some of the resistance genes. It may also be due to using different surfactants in the formulation of microemulsions or using different antibacterial evaluation techniques. Generally speaking, the surfactant used in microemulsion formulation can play a prominent roll on the antibacterial activities of the tested antimicrobial oils. Positively charged surfactant（cationic）can easily be adhering to the negatively charged bacterial cell wall via electrostatic attraction, making the antibacterial agent more effective. Such interaction is absent in the case of nonionic surfactants, as the case in the current study. However the role of nonionic surfactant is also thought to be crucial in contributing to the observed antibacterial activity. These surfactants tend to form micelles in water which incorporate （host）the antibacterial oils and form water-based microemulsions. The particle size of the antimicrobial（EO and eugenol）in the microemulsions formulated in the current study was ∼8.00 nm. That nano-size can potentially increase the passive cellular absorption mechanisms of the antibacterial, therefore reducing mass transfer resistances and increasing antimicrobial activity. That was supported by previous studies which showed that nonionic surfactantbased microemulsions improved the antibacterial properties of eugenol 55−57）. However in the current study we cannot confirm or deny that statement concerning N. sativa EO until that oil was tested in its neat versus its microemulsified states. One may also consider the potential diminish of the antimicrobial activity of microemulsions due to the physical interactions between the antimicrobial oil and the nonionic surfactant that used in the formulation. For instance, some nonionic surfactants especially from the Tween family can 229
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bind to the polar bioactive group of some essential oil constituents by forming hydrogen bonds. That can lead to deactivation and partial loss of EO antibacterial activity58−60）. It worth also noting that, the water soluble fraction of the EOs and their constituents（ via hydrogen bonds）in equilibrium with the amount incorporated in the surfactant micelles can also contribute to the antibacterial activity of emulsions61）. That is mainly because bacteria normally lives and proliferates in water. Therefore the extent of partition of the antibacterial EO in water along with its colloidal nano-size in microemulsion and its interactions with the surfactant can determine the over all antibacterial properties of the antimicrobial microemulsions. On the other hand, the role of the surfactant is ignored in discussing the antibacterial activity of Ceftriaxone（the synthetic antibiotic）due to its hydrophilic nature. That property enabled direct re-constitution of Ceftriaxone in water without the need of formulation in surfactant-aided colloidal system as microemulsions.
4 CONCLUSIONS This study is the first report that revealed the antibacterial activity of N. sativa EO microemulsion against different pathogenic bacteria. This activity resembled that of the synthetic antibiotic Ceftriaxone against S. typhimurium at 300.0 μg/well and surpassed Ceftriaxone at higher concentrations. In addition the EO microemulsion showed antibacterial activity comparable to that of Ceftriaxone against E. coli at 500.0 μg/well. The EO microemulsion was more effective than eugenol microemulsion against all tested pathogens except for P. aeruginosa. The revealed chemical composition of N. sativa EO can be imitated using synthetic ingredients to satisfy mass production if natural resources of that EO are not sufficient. In such case, more selective control over the chemical composition can be granted to secure reproducible antimicrobial activity and regular supply. A complementary work is planed by our team to investigate the combination of N. sativa EO microemulsion with other synthetic antibiotics in the same formula for potential enhanced antimicrobial activity. In addition, the validity of that EO microemulsion as a preservative in real food system is also a subject of future investigation.
Conflict of Interest The authors declare that there is no any potential source of conflict of interest that editors may consider relevant to manuscript.
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Analysis and Antibacterial Activity of Nigella sativa Essential Oil Formulated in Microemulsion System Hamdy A. Shaaban1, Zainab Sadek2, Amr E. Edris1＊ and Amal Saad-Hussein3 1
Aroma & Flavor Chemistry Department, National Research Center, Cairo, Egypt Dairy Department, National Research Center, Cairo, Egypt 3 Environmental & Occupational Medicine Department National Research Center, Cairo, Egypt 2
Abstract: The Essential oil (EO) of Nigella sativa (black cumin) was extracted from the crude oil and the volatile constituents were characterized using gas chromatographic analysis. The EO was formulated in water-based microemulsion system and its antibacterial activity against six pathogenic bacteria was evaluated using the agar well diffusion method. This activity was compared with two other well known biologically active natural and synthetic antimicrobials namely eugenol and Ceftriaxone®. Results showed that N. sativa EO microemulsion was highly effective against S. aureus, B. cereus and S. typhimurium even at the lowest tested concentration of that EO in the microemulsion (100.0 μg/well). Interestingly, the EO microemulsion showed higher antibacterial activity than Ceftriaxone solution against S. typhimurium at 400.0 μg/well and almost comparable activity against E. coli at 500.0 μg/well. No activity was detected for the EO microemulsion against L. monocytogenes and P. aeruginosa. Eugenol which was also formulated in microemulsion was less effective than N. sativa EO microemulsion except against P. aeruginosa. The synthetic antibiotic (Ceftriaxone) was effective against most of the six tested bacterial strains. This work is the first report revealing the formulation of N. sativa EO in microemulsion system and investigating its antibacterial activity. The results may offer potential application of that water-based microemulsion in controlling the prevalence of some pathogenic bacteria. Key words: antibacterial activity, Nigella sativa, essential oil, eugenol, microemulsion, Ceftriaxone 1 INTRODUCTION Essential oils（EOs） are natural mixtures of volatile compounds secreted as by-products by many aromatic and medicinal plants. These oils act as insect attracting agents for pollination and also as a defense line against microbial attack. EOs were first used as perfuming and flavoring agents then their inherent antimicrobial activity was discovered. Different investigations on the application of EOs against pathogenic bacteria, food spoilage and film forming microorganisms have been reported1−4）. The antimicrobial properties of EOs against pathogens were studied in different sanitizing applications including sanitization of wounds5） and surfaces6）, disinfection of animal houses7） and sewage grey water8）. The promising results of these investigations along with many others warranted EOs for consideration as potential candidates for application as adjuvant for the synthetic antibiotics. EOs can enhance the therapeutic efficiency of these antibiotics through a synergistic mechanism9, 10）. Combination of antibiotics with some
EOs can also reduce the well known antibiotic resistance in multidrug resistant bacteria11）. Interestingly, it was found that formulation of EOs as nanoparticles in water-based delivery systems can enhance their antimicrobial activity compared with the neat unformulated form of these oils12, 13）. The mechanisms that clarify the role of nanoparticle in boosting the antimicrobial activity of EOs were reviewed in details14）. The above mentioned literature included antimicrobial evaluations of wide varieties of EOs isolated from different aromatic plants. However, we noticed some limitation of conclusive data regarding the antibacterial activity of Nigella sativa EO especially after formulation in microemulsion. The seeds of that herbaceous plant（family Ranunculaceae）are the part that bears the EO. The antimicrobial evaluation of the seeds and their solvent extracts were studied15−18）. Specific targeting on the antimicrobial evaluation of the pure EO fraction of N. sativa has also been approached19）. However that study did not address
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Correspondence to: Amr E. Edris, Aroma & Flavor Chemistry Department National Research Center El Behose Street, Dokki, 12622 Cairo, Egypt E-mail: [email protected] Accepted September 27, 2014 (received for review August 10, 2014)
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some important issues that usually correlated with the antimicrobial activity of N. sativa EO. These include the chemical composition and the percentage of the major volatile constituents of that EO especially thymoquinone （TQ） . On the other hand, some other antimicrobial evaluations considered and correlated the chemical composition of the EO to the reported activity20−22）. However, these investigations did not include some important pathogenic bacteria in their evaluations like Bacillus cereus, Listeria monocytogenes, and Salmonella typhimurium. Most importantly, no studies were found, to our knowledge, evaluating the antibacterial activity of N. sativa EO as nanoparticles formulated in water-based microemulsion delivery system. From the above mentioned, the following investigation was dedicated to provide more information on the antibacterial activity of that EO microemulsion which may not be addressed in previous studies. This included extraction and analysis of the EO followed by formulation in waterbased microemulsion system and finally evaluation of the overall antibacterial activity of the formulated EO against different pathogenic bacteria. Moreover, the antibacterial activity of the EO microemulsion was compared with that of a well know plant-based phenolic antimicrobial model namely eugenol which was formulated also in microemulsion. A synthetic antibiotic model （Ceftriaxone） was used as a reference to help in ranking the antimicrobial activity of N. sativa EO microemulsion among the natural（eugenol） and the synthetic （Ceftriaxone） antimicrobial models.
2 EXPERIMENTAL 2.1 Materials Packages of N. sativa crude oil encapsulated in hard gelatin capsules were purchased from a local pharmacy in Cairo, Egypt. Each capsule contains 450.0 mg of a 100.0％ pure crude oil extracted from the seeds of N. sativa by cold expression. No other additives are included in the capsules, as indicated by the manufacturing company （Pharco Pharmaceuticals, Cairo, Egypt） . The synthetic antibiotic Ceftriaxone® sodium powder （containing 1000 mg Ceftriaxone） was also purchased from local pharmacy in Cairo, Egypt. The drug is produced by SmithKline Beecham Egypt under license from Sandoz. ® sorbitane Eugenol 99.0％ and Tween 20（polyoxyethylene mono-laurate）were purchased from Sigma-Aldrich Chemical Co. （St. Louis, MO, USA） 2.2 Extraction of the pure EO fraction Sufficient numbers of capsules bearing N. sativa whole crude oil（which is a natural mixture of fixed oil and EO）, were broken and evacuated into a beaker. The pure EO fraction was extracted from the crude oil by the hydro-dis-
tillation method as previously indicated 23）. In brief, the crude oil was mixed with distilled water（1:5 w/v）and distilled for three hours using Clevenger-type apparatus. The cup that receives the pure EO fraction in the side arm of the apparatus was wrapped with aluminum foil to protect the EO from light that can dimerize its main constituent thymoquinone into di-thymoquinone. At the end of the distillation process, the EO was collected, weighed and its content（yield percent）relative to the weight of total crude oil was calculated as mean of two extraction processes. 2.3 Gas chromatographic analysis （20.0 μl） was diluted in 1.0 ml diethyl ether N. sativa EO in a glass vial. Then 2.0 μl of this mixture were injected （at a split ratio 10:1）into a Perkin Elmer XL GC equipped with a FID. A fused silica capillary column（60 m×0.32 mm× 0.25 μm）coated with DB-5（5％ phenyl, 95％ methyl polysiloxane）was used to separate the different EO components. The oven temperature was programmed from 50℃ to 230℃ at a rate of 3℃/min. The injector and detector temperatures were 230℃ and 250℃ respectively. Helium was used as carrier gas at a flow rate of 1.0 ml/min. The volatile oil constituents were quantified as percent of the total detected peak areas after flame ionization detection. All values were mean of two injections from two different extractions±SD. The available authentic samples were used to reveal the identity of N. sativa EO constituents by matching their retention times with those of N. sativa EO constituents after running on GC under the same conditions. 2.4 Gas chromatographic-mass spectroscopic analysis GC-MS analysis was conducted using a Hewlett-Packard 5972 GC-MS system equipped with the same column and conditions used as in GC analysis. The ionization voltage was 70 eV and the ion source temperature was 170℃. The scan range was from 29 to 300 AMU at 2.76 scans per second. Components identity that was previously revealed by running authentic samples in GC analysis was verified by automatic matching of their fragmentation patterns（m/ z）with standard components stored in the electronic mass spectral library（NIST: National Institute of standards and Technology）that was built-in the GC-MS soft ware. 2.5 Formulation of N. sativa EO and eugenol in waterbased microemulsions Surfactant solution was prepared by dissolving the nonionic surfactant（Tween 20®）in de-ionized water at 5.0％ （w/w）using magnetic bar. The clear solution was divided equally by weight among different glass vials wrapped with aluminum foil to protect from light. Different volumes of the hydrophobic N. sativa EO and eugenol were titrated separately into each vial to end up with 10 concentrations for both EO and eugenol, ranging between 0.2％ - 2.0％
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with a concentration gradient of 0.2％. This percentage was equivalent to 2.0 mg - 20.0 mg EO or eugenol/ml microemulsion with concentration gradient 2.0 mg. All vials were vortexed for 1 minute to mix the ingredients and hasten the formation of microemulsion then left in dark at 27℃±1 to equilibrate. All formulations were also made in duplicates. 2.6 Formulation of Ceftriaxone in aqueous solution Unlike the hydrophobic EO and eugenol, Ceftriaxone ® hydrophilic powder was re-constituted directly in distilled water to give 1.0％ aqueous solution（i.e. 10 mg/1 ml water）. Then serial dilutions were made from that stock solution as required for utilization in the antibacterial assay. Thus there was no need to formulate Ceftriaxone in water-based microemulsion as it is a water-soluble antimicrobial. 2.7 Verification of microemulsion formation Evidence of microemulsion formation for N. sativa and eugenol was based on the clear and transparent appearance of the formulations after the end of equilibration time which reached 4 weeks. The non-birefringent texture under polarized light microscope and the small hydrodynamic particle size（r）which did not exceed 100.0 nm was also taken as an evidence of microemulsion formation. 2.8 Particle size measurement The particle size of microemulsions was measured using the dynamic light scattering instrument Zetasizer （Nano-ZS model ZEN3600, Nanoseries, Malvern Instruments, UK）. Measurements were done at 27℃, with a fixed angle of 173° . The measurements are based on the Brownian motion of the hydrated particles, thus it provides information on the hydrodynamic diameter（nm）of the microemulsion particles. Sizes quoted are the z-average mean of the microemulsion hydrodynamic diameter（nm）obtained from 10 measurements （2 replicate×5 measurements each） . Before measurement, the samples were filtered through 0.20 μm single use syringe filter unit（Minisart®, Sartoius Stedium Biotech GmbH Germany） to remove impurities. 2.9 Antibacterial evaluation 2.9.1 Microorganisms and cultures The tested microorganisms were provided from the culture collections of the Microbiological Department National Research Center（NRC）Dokki, Giza, Egypt. These include three strains of Gram-positive bacteria Staphylococcus aureus（ ATCC 43300）, Bacillus cereus（ ATCC 11778） , Listeria monocytogenes （ATCC 35152） , and three strains of Gram-negative bacteria Salmonella typhimuri（ATCC 13311） , Escherichia coli （ATCC 27325） , Pseuum domonas aeruginosa （ATCC 27853） .
2.9.2 Antibacterial assay The antimicrobials N. sativa EO, eugenol and Ceftriaxone formulated in their corresponding delivery systems （see formulation section）were evaluated against the six previously mentioned pathogenic bacteria. The evaluation was performed at five chosen concentrations of the three antimicrobials: 0.2％, 0.4％, 0.6％, 0.8％ and 1.0％, unless otherwise stated. These percentages are equivalent to 2.0 mg antimicrobial/1.0 ml formula, 4.0 mg/ml, 6.0 mg/ml, 8.0 mg/ml and 10.0 mg/ml, respectively. The agar well diffusion method24） was employed for the determination of antibacterial activities of the three formulated antimicrobials. In details, 0.1 ml of the diluted inoculums（107 CFU/ml） of test organism was spread on tryptone soy agar（TSA） plates. Wells of 5 mm diameter were punched into the agar medium and filled with 50 μl of each concentration of the three evaluated antimicrobial formulas. This volume delivered 100.0 μg, 200.0 μg, 300.0 μg, 400.0 μg and 500.0 μg of each antimicrobial（EO, eugenol and Ceftriaxone）per well. The plates were incubated for 18 h at 37℃. Antimicrobial activity was evaluated by measuring the zone of inhibition （mm）against the tested organisms. The evaluation was conducted twice and each time comprised three replicates for each concentration. 2.10 Statistical analysis Statistical analysis was done through SPSS version 18.0. Quantitative data were represented in form of mean±standard deviation（ SD）. Analysis of Variance（ ANOVA）was used in the analysis of the results. The post hoc Duncan was used to make pair wise comparisons using a stepwise order of comparisons identical to the order used by the Student-Newman-Keuls test, but sets a protection level for the error rate for the collection of tests, rather than an error rate for individual tests. The level of significant was considered at p＜0.05.
3 Results and Discussions 3.1 Analysis of N. sativa EO The content of the pure N. sativa EO fraction after extraction from the whole crude oil using the hydro-distillation method was 2.2±0.1 wt ％. This percentage is considered to be much higher than other samples of N. sativa that yield 0.11-1.8 wt ％ EO relative to the crude oil23）. The genetic diversity of the seeds is among the most important factors that affect the quality and content of the EO25）. Table 1 showed the main constituents of the EO as re（TQ） vealed by the GC and GC-MS analysis. Thymoquinone was the major constituent of the EO（52.6％）followed by p-cymene（25.8％）, α-thujene（10.5％）, β-pinene（3.0％） and α-pinene（2.8％）. Other minor constituents were also 225
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Table 1 Main constituents of N. sativa EO as revealed by GC and GC-MS analysis. Retention time (min)
Area %#
α-thujene*
Component
14.3
10.50±0.7
93 (100.0%), 77, 91, 92, 41, 39, 27, 79, 94, 136
α-pinene*
14.6
2.8 ±0.2
93 (100.0%), 91, 92, 39, 77, 41, 79, 27, 53, 29
sabinene
16.3
0.5 ±0.07
93 (100.0%), 41, 91, 77, 79, 39, 27, 69, 94, 43
β-pinene*
16.6
3.0 ±0.3
93 (100.0%), 41, 69, 39, 91, 77, 79, 27, 92, 53
O-cymene
18.3
0.1 ±0.02
119(100.0%), 91, 134, 117, 77, 65, 115, 39, 120, 51
p-cymene*
18.7
25.8 ±0.7
limonene*
19.0
1.1 ±0.1
68 (100.0%), 93, 67, 94, 39, 92, 107, 53, 79, 136
linalool*
22.3
0.5 ±0.05
71 (100.0%), 93, 55, 43, 41, 69, 80, 121, 67, 39
iso-3-thujanol
22.9
1.36±0.2
43 (100.0%), 55, 41, 93, 95, 81, 67, 79, 121, 69
thymoquinone*
29.6
52.6 ±1.9
α –longipinene
33.4
0.3 ±0.03
119 (100.0%), 105, 133, 93, 107, 91, 161, 120, 204, 121
longifolene
35.8
1.5 ±0.1
161 (100.0%), 94, 91, 93, 107, 105, 95, 79, 55, 109
Total
Mass fractions of each component (m/z)**
119(100.0%), 134, 91,120, 117, 41, 77, 39, 65, 115
164 (100.0%), 121, 136, 93, 149, 39, 40, 53, 77, 91
98.96
#: after GC analysis via flam ionization detection * Component identity was confirmed by matching retention times with authentic samples, beside the GC-MS identification. ** Mass fractions of each component were arranged in descending order according to the % abundance. identified and illustrated in the table. This composition authenticated and confirmed the identity of N. sativa crude oil in the gelatin capsules because high p-cymene and TQ percentages in the EO are considered as markers for the sativa species. p-cymene and TQ can reach only 0.0-0.5％ and 0.0-0.1％, respectively in the EO of other Nigella species like for instance Nigella orientalis, N. damascene and N. arvensis26−28）. 3.2 Formulation of microemulsions After the characterization of N. sativa EO, five concentrations of this oil and the pure eugenol were formulated separately in water-based microemulsion systems with the aid of surfactant（Tween 20）. Micro-emulsification technique is simple and economically feasible on small as well as large scale for solubilizing hydrophobic bioactive oils in water-based environment as nano-particles. This process is spontaneous and driven by the reduction of the system’ s free energy due to interaction of the hydrophobic solubilizates （N. sativa EO and eugenol） with the surfactant molecules at certain energetically favorable orientation. One should consider that formulation of both EO and eugenol in water-based micoemulsion is necessary due to the hydrophobic nature of these antimicrobials which prevent direct dissolution in water. On the other hand, the synthetic antibiotic Ceftriaxone was re-constituted directly in
Fig. 1 P article size distribution of Nigella EO and eugenol microemulsions measured at their maximum concentrations (1.0% and 0.9%), respectively. water due to its hydrophilic nature. Figure 1 showed that the particle size of the EO and eugenol in the microemulsion formulations was 8.7±0.1 nm and 8.9±0.1 nm, respectively with mono-modal size distribution. Examination of the different formulation under the polarized light microscope revealed no birefringence. Visual observation showed clear and transparent
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Fig. 2 C hemical structures of thymoquinone and eugenol. formulation with water-like flowability. These data confirmed the formation of typical water-based microemulsion systems. Both microemulsions were found to be physically stable when stored at 37℃ for 24.0h before the evaluation of their antibacterial activity. They were also physically stable towards different freeze-thaw cycles and toward centrifugation at 5000 rpm for 30 min which confirmed their kinetic and thermodynamic stability as microemulsions. It was interesting to note during formulation that eugenol took only ~ 30.0 minutes to equilibrate into a clear transparent microemulsion while N. sativa EO needed longer time that can reach 4.0 week to equilibrate especially at high concentration like 1.0％（i.e. 10.0 mg EO/ml microemulsion）. The reason of that observation lies in the chemical structure of eugenol that contains hydrophilic and hydrophobic-like parts resembling the surfactant structure（ Fig. 2）. Due to that structure, eugenol can arrange itself at the interfacial film along with the original surfactant（Tween 20）. That arrangement makes the original surfactant more efficient and facilitates the formation of microemulsion29）. That advantage is not available for N. sativa EO which is a complex mixture of volatile components of different chemical structures and polarities. One of the liabilities of microemulsion formulation is the low pay load of the bioactive oil that can be incorporated in the water-surfactant solution to form microemulsion. Thus it was important to determine the maximum oil concentration that can be incorporated into the microemulsion before evaluating the antibacterial activity. For N. sativa EO that concentration was found to be 1.4 ％（i.e. 14 mg EO/ ml microemulsion）under the formulation conditions adopted in the current study. However at that concentration, crystals of TQ, which is the major constituent of the EO（52.6％）, were precipitated out from the microemulsion. Therefore the upper limit of N. sativa EO used for assessing the antibacterial activity in the microemulsion was set at 1.0％ （10 mg EO/ml microemulsion, or 500.0 μg/ well）. This concentration proved to be the safe margin for keeping the integrity of the EO constituents （including TQ） as being totally incorporated in the microemulsion. On the
other hand concerning eugenol, the maximum pay load in the microemulsion was found to be 0.9％（9.0 mg eugenol/ under the same formulation conditions. ml microemulsion） Trials to increase that load to reach 1.0％ in order to match that of N. sativa EO was unsuccessful as it turned the transparent eugenol microemulsion into a cloudy dispersion of macroemulsion. The particle size of eugenol in that macroemulsion was out of the microemulsion size range （＞ 100 nm）as revealed by the visual cloudy appearance and the presence of large particles under the light microscopic. 3.3 Antimicrobial evaluation Table 2 showed the antimicrobial activity of the three tested formulas namely, EO microemulsion, eugenol microemulsion and Ceftriaxone solution against some pathogenic bacteria. The table showed that the EO microemulsion was more antibacterial active than eugenol microemulsion at all concentrations against S. aureus, B. cereus and S. typhimurium, while eugenol was more effective against P. aeruginosa. The activity of eugenol microemulsion against P. aeruginosa reached its climax at 500.00 μg/well at which there was no significant difference between the antibacterial activity of eugenol microemulsion（40.0 mm）and that of Ceftriaxone（40.3 mm）. Interestingly, there was no significant difference between the antimicrobial activity between N. sativa EO microemulsion（18.0 mm）and Ceftriaxone（19.3 mm）against S. typhimurium at 300.0 μg/ well. In addition, the EO microemulsion showed even higher antibacterial activity than Ceftriaxone against S. typhimurium at 400.0 μg and 500.0 μg. The EO microemulsion also showed close antimicrobial activity（30.3 mm）to Ceftriaxone（31.3 mm）against E. coli at 500.0 μg. From Table 2 it is evident that the EO microemulsion of N. sativa at high concentrations（400.0 μg/well and 500.00 μg/well）possesses antibacterial activity against four out of six of the tested pathogens. The EO microemulsion showed its highest antibacterial activity at the lowest concentration （100.0 μg EO/well）against S. aureus and B. cereus, respectively. E. coli was more resistant to EO microemulsion up to 300.0 μg/well, and then as the concentration of EO increased to 400.0 μg/well the antimicrobial activity also increased until reaching maximum（30.3 mm）at 500.0 μg/ well. On the contrary, L. monocytogenes and P. aeruginosa were the most resistant bacterial strains toward EO microemulsion treatments at all concentrations. The highest antimicrobial activity for EO microemulsion folat 500.0 μg/well was found against S. aureus（33.7 mm） lowed by E. coli（30.3 mm）, S. typhimurium（26.5 mm） and B. cereus（21.3 mm）, respectively. Table 2 also showed that eugenol microemulsion possessed high antibacterial activity against P. aeruginosa at all concentrations. Eugenol microemulsion was not effective at all concentrations against B. cereus and L. monocytogenes. However, it started to show concentration depen227
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Table 2 Effect of the three tested antibacterial formulas against some pathogenic bacteria. Concentration*
Antibacterial formulas EO microemulsion
100.0 μg/well
Eugenol microemulsion Ceftriaxone solution P-value EO microemulsion
200.0 μg/well
Eugenol microemulsion Ceftriaxone solution P-value EO microemulsion
300.0 μg/well
Eugenol microemulsion Ceftriaxone solution P-value EO microemulsion
400.0 μg/well
Eugenol microemulsion Ceftriaxone solution P-value
500.0 μg/well
S. aureus
E. coli
B. cereus
L.monocytogenes
P. aeruginosa
S. typhimurium
Inhibition zone (mm) Mean
SD
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Mean
SD
15.7
0.6
0.0a
0.0
14.7
0.6
0.0a
0.0
0.0
0.0
8.0
1.7
0.0
0.0
0.0a
0.0
0.0
0.0
0.0a
0.0
16.0
1.0
0.0
0.0
39.67
0.6
24.0
1.0
27.0
1.7
16.7
1.5
34.0
1.7
16.7
1.5
P< 0.0001
P< 0.0001 a
P< 0.0001
P< 0.0001 a
P< 0.0001
P< 0.0001
19.7
2.1
0.0
0.0
16.3
1.5
0.0
0.0
0.0
0.0
16.0
0.0
0.0
0.0a
0.0
0.0
0.0
0.0a
0.0
21.3
1.5
0.0
0.0
41.7
0.6
25.7
1.2
28.3
1.5
17.7
1.5
36.0
1.0
18.0
1.0
P< 0.0001
P< 0.0001
2.3
0.0a
0.0
0.0
a
0.0
0.0
43.3
0.6
27.3
0.6
25.3
P< 0.0001
0.0
P< 0.0001
P< 0.0001
P< 0.0001
1.0
0.0a
0.0
0.0
0.0
a
29.7
1.2
18.7
18.0
P< 0.0001
0.0
P< 0.0001 0.0
0.0
1.0
P< 0.0001 18.0a
1.7
0.0
26.0
1.4
0.0
0.0
1.5
37.3
0.6
19.3a
1.2
P< 0.0001
P< 0.0001
P< 0.0001
33.0
1.0
25.3
1.5
19.6
1.5
0.0a
0.0
0.0
0.0
22.3
0.6
7.7
0.6
13.5
0.7
0.0
0.0
0.0a
0.0
32.7
1.2
12.3
0.6
44.7
0.6
29.0
1.0
32.0
1.0
20.0
1.0
39.3
0.6
21.7
1.2
P< 0.0001
P< 0.0001
P< 0.0001
P< 0.0001
P< 0.0001
P< 0.0001
EO microemulsion
33.7
1.5
30.3
1.5
21.3
1.5
0.0a
0.0
0.0
0.0
26.5
0.7
Eugenol microemulsion
12.7
0.6
15.7
1.2
0.0
0.0
0.0a
0.0
40.0a
1.0
16.3
0.6
Ceftriaxone solution
46.0
1.0
31.3
1.6
34.7
0.6
21.0
2.0
40.3a
0.6
23.7
2.1
P-value
P< 0.0001
P< 0.0001
P< 0.0001
P< 0.0001
P< 0.0001
P< 0.0001
* The content of the antimicrobial agent (μg) which present in 50.0 μl of each antimicrobial formula /well a : Means with the same letter are not significantly different. The highest tested concentration of eugenol in the microemulsion was 0.9% (450 μg/well) due to formulation restriction (as indicated previously in the results and discussion section).
dent antibacterial activities above 300.0 μg/well against S. aureus, E. coli and S. typhimurium. The inhibition zone of P. aeruginosa was still significantly larger than that of these three strains at concentration above 300.0 μg/well. From the table it is also clear that Ceftriaxone solution had high antibacterial active against all bacterial strains at all concentrations. However, it was more effective against S. aureus and P. aeruginosa compared with L. monocytogenes and S. typhimurium at the lowest and highest tested concentrations. It is impotent to note that the control sample of the surfactant and water at 5.0％ did not show any antibacterial activities. These results in general indicated a selective and high antibacterial activity of N. sativa EO. This activity is thought to be due to the high content of TQ which constitutes 52.6％ of the EO composition（Table 1）. This compound is a phyto-quinone present only in the EO of N. sativa. One of the most important antimicrobial characteristics of TQ is its ability to modify the resistance of bacteria to antibiotics by acting as efflux pump inhibitor30）. Efflux pumps are proteins in the cell membrane of the bacteria that extrude the antimicrobial agents outside the cell31）. Thus multi-drug resistant bacterial strains can arise 32）
which threaten the lives of patients infected with these strains. TQ was efficient in inhibiting several multi-drug resistant pathogenic bacteria19）. It can kill antibiotic resistant S. aureus at 6.0 μg/ml33）. TQ also has anti-biofilm properties against some Gram-positive and Gram-negative bacteria34）. Beside literature evidences that points out to TQ as the major antibacterial active principle of N. sativa EO one should bear in mind the potential synergistic effect of TQ with other component（s）that constitute the EO. For instance, p-cymene which constitutes 25.8％ of the EO composition may act as a potential synergistic agent. This terpenic hydrocarbon was previously found to act synergistically with carvacrol against B. cereus in rice35） and against Escherichia coli O157:H7 in un-pasteurised apple Juice36）. p-Cymene also showed a high affinity for microbial membranes however it does not affect the membrane permeability but it may decrease its enthalpy and melting temperature37）. In addition, this compound can perturb the microbial membranes, causing them to expand thus affecting the membrane potential of the intact cells38）. Synergism can also be rationalized in some cases by the protection of the major antimicrobial principle of a given EO from degradation by the microbial enzymes or by con-
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tribution in altering the multi-drug resistance mechanism39）. To our knowledge, there is no data so far revealing the potential synergistic activity between p-cymene and TQ which could be a subject for a future research work. Despite this evidence-based antibacterial activity of N. sativa EO microemulsion, it was challenging to find it not effective against L. monocytogenes and P. aeruginosa at all concentrations （Table 2） . The relatively simple structure of the cell wall of L. monocytogenes as Gram-positive bacteria is supposed to make it vulnerable to a wide range of antibacterial agents. However the antimicrobial resistance of L. monocytogenes is not an unusual biological event. This phenomena was previously reported in different studies from samples taken from dairy farms 40）, seafood products41）, and from ready-to-eat meat products42）. The resistance of L. monocytogenes originates from some antimicrobial resistance genes that limit the number of the effective anti-listerial agents, as revealed from the last three references. Details of the resistance mechanism of L. monocytogenes were reviewed elsewhere43）. P. aeruginosa was found to be another resistant bacterium to N. sativa EO microemulsion. The complex cell envelope of this organism resembles that of other Gram-negative bacteria which consist of polysaccharide, protein, lipopolysaccharide, and lipid components 44）. It is well known that P. aeruginosa possessed a great adaptability and metabolic versatility. It has adaptive resistance toward some antibiotics45, 46） and biocides47）. Adaptive resistance refers to reversible refractoriness to the bactericidal action of an antibiotic following first exposure. This phenomenon has been well documented in P. aeruginosa following exposure to an aminoglycoside48）. Thus the potential reasons for resistance to antimicrobials are a combination between the inherent multidrug efflux pumps with chromosomallyencoded antibiotic resistance genes49）and the barrier function of the outer complex cell membrane. Another acquired resistance factor may also develop by mutation in chromosomally-encoded antibiotic resistance genes50）. A detailed contribution explaining the mechanism of antibiotic resistance in P. aeruginosa is also available elsewhere51）. Interestingly, with the presence of all the above mentioned resistance mechanisms against antibacterial agents, eugenol microemulsion was found to be highly effective against P. aeruginosa than N. sativa EO at all concentrations（ Table 2）. The activity of eugenol microemulsion against that pathogen was almost the same as that of Ceftriaxone at the highest concentration（ 500.0 μg/well）. Another study also reported high antibacterial activity of clove bud ethanolic extract, as a rich source of eugenol, against P. aeruginosa52）. However, the incidence of antimicrobial sensitivity for that organism varied depending on the isolate strain and source of isolation （human, animal or environment）. This may explain why two strains of P. aeruginosa exhibited intrinsic tolerance to different plant-
based volatiles including pure eugenol53）. The disrupting effect of eugenol on the cytoplasmic membrane of the bacterial cell54） may justify its observed effectiveness against P. aeruginosa in our study. Another reason may include a certain affinity between the surfactant（Tween 20）that associate with the active phenolic group of eugenol in the microemulsion, and the cell membrance of P. aeruginosa. That affinity can induce better adsorption of the surfactant/eugenol complex to the surface of the bacterial cell which in turn will increase its local concentration at that point. However, a conclusive justification of the enhanced antibacterial activity of eugenol microemulsion against P. aeruginosa in our study is certainly challenging. That is particularly due to the lack of any antibacterial activity of eugenol microemulsion against L. monocytogenes and B. cereus at all concentrations（Table 2） . In contrast to this result, eugenol microemulsion was found in other studies effective against L. monocytogenes at concentrations close to that used in the current study55−57）. This contradiction may originate from using different strain or isolate of L. monocytogenes that lack some of the resistance genes. It may also be due to using different surfactants in the formulation of microemulsions or using different antibacterial evaluation techniques. Generally speaking, the surfactant used in microemulsion formulation can play a prominent roll on the antibacterial activities of the tested antimicrobial oils. Positively charged surfactant（cationic）can easily be adhering to the negatively charged bacterial cell wall via electrostatic attraction, making the antibacterial agent more effective. Such interaction is absent in the case of nonionic surfactants, as the case in the current study. However the role of nonionic surfactant is also thought to be crucial in contributing to the observed antibacterial activity. These surfactants tend to form micelles in water which incorporate （host）the antibacterial oils and form water-based microemulsions. The particle size of the antimicrobial（EO and eugenol）in the microemulsions formulated in the current study was ∼8.00 nm. That nano-size can potentially increase the passive cellular absorption mechanisms of the antibacterial, therefore reducing mass transfer resistances and increasing antimicrobial activity. That was supported by previous studies which showed that nonionic surfactantbased microemulsions improved the antibacterial properties of eugenol 55−57）. However in the current study we cannot confirm or deny that statement concerning N. sativa EO until that oil was tested in its neat versus its microemulsified states. One may also consider the potential diminish of the antimicrobial activity of microemulsions due to the physical interactions between the antimicrobial oil and the nonionic surfactant that used in the formulation. For instance, some nonionic surfactants especially from the Tween family can 229
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bind to the polar bioactive group of some essential oil constituents by forming hydrogen bonds. That can lead to deactivation and partial loss of EO antibacterial activity58−60）. It worth also noting that, the water soluble fraction of the EOs and their constituents（ via hydrogen bonds）in equilibrium with the amount incorporated in the surfactant micelles can also contribute to the antibacterial activity of emulsions61）. That is mainly because bacteria normally lives and proliferates in water. Therefore the extent of partition of the antibacterial EO in water along with its colloidal nano-size in microemulsion and its interactions with the surfactant can determine the over all antibacterial properties of the antimicrobial microemulsions. On the other hand, the role of the surfactant is ignored in discussing the antibacterial activity of Ceftriaxone（the synthetic antibiotic）due to its hydrophilic nature. That property enabled direct re-constitution of Ceftriaxone in water without the need of formulation in surfactant-aided colloidal system as microemulsions.
4 CONCLUSIONS This study is the first report that revealed the antibacterial activity of N. sativa EO microemulsion against different pathogenic bacteria. This activity resembled that of the synthetic antibiotic Ceftriaxone against S. typhimurium at 300.0 μg/well and surpassed Ceftriaxone at higher concentrations. In addition the EO microemulsion showed antibacterial activity comparable to that of Ceftriaxone against E. coli at 500.0 μg/well. The EO microemulsion was more effective than eugenol microemulsion against all tested pathogens except for P. aeruginosa. The revealed chemical composition of N. sativa EO can be imitated using synthetic ingredients to satisfy mass production if natural resources of that EO are not sufficient. In such case, more selective control over the chemical composition can be granted to secure reproducible antimicrobial activity and regular supply. A complementary work is planed by our team to investigate the combination of N. sativa EO microemulsion with other synthetic antibiotics in the same formula for potential enhanced antimicrobial activity. In addition, the validity of that EO microemulsion as a preservative in real food system is also a subject of future investigation.
Conflict of Interest The authors declare that there is no any potential source of conflict of interest that editors may consider relevant to manuscript.
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